The present invention relates generally to recombinant methods and materials for effecting the microbial production of useful polypeptides and more particularly to novel DNA plasmids useful in securing exceptionally high levels of expression of exogenous genes in E.coli host cells.
Numerous attempts have been made to optimize the extent to which exogenous genes are subject to expression in microbial host cell populations such as E.coli, B.subtilis, yeast and other microbial cell types. Among the initially more promising developments in the optimization of E.coli expression of "foreign" genes was isolation of the so-called "temperature dependent", "temperature sensitive", or "runaway mutant" forms of circular DNA plasmid. See, generally, Uhlin, et al., Gene, 6, pp. 91-106 (1979); Uhlin, et al., J.Bacteriol., 148, pp. 386-390 (1981); Uhlin, et al., U.K. Pat. No. 1,557,774. These autonomously-replicating plasmids could generally be maintained in E.coli host cells in moderate copy numbers when the cells were cultured at a "permissive" temperature below 34.degree. C., e.g., at about 30.degree. C. Upon elevation of culture temperatures to a "non-permissive" level of 37.degree. C. and above, inherent controls on autonomous replication of the plasmid within the hose were lost and replication of the plasmid was said to "run away" until quite substantial numbers of copies of the plasmid were present in the cell.
The potential utility of such temperature sensitive runaway mutant plasmids in recombinant methods for securing E.coli expression of exogenous gene products was manifest. Because it is often the case that expression of exogenous genes results in formation of products which are toxic or otherwise detrimental to cell metabolism and because yields of exogenous gene products are often diminished by host cell degradation of the foreign polypeptides over a period of time, it was believed that overall yields of desired products could be optimized by delaying replication of recombinant plasmids until host cells had reached their maximum cell culture densities in the fermentor. In this way large scale DNA transcription and mRNA translation required for host expression of a desired adventitious gene could be temporally regulated to occur at a time close to final product harvest, when host cell detriment and product degradation would have minimal effects of overall product yields.
The first of the temperature sensitive runaway mutant plasmids to be isolated, however, suffered numerous drawbacks which made them unattractive as potential expression vectors for use in securing large scale microbial synthesis of commercially significant proteins. First of all, the plasmids were quite large (averaging around 10 or 12 Kb in size) and their runaway replication in host cells thus constituted a substantial energy drain. This difficulty was alleviated in part upon the development of smaller sized, "mini-plasmids" such as the 4.6 Kb pKN402 (Deutsche Sammlung von Microorganismen, "DSM" Accession No. 1228) but was exacerbated even for the mini plasmids upon insertion of one or more "marker" genes coding for a protein product indicative of plasmid maintenance in the host (e.g., a gene for coding for .beta.-lactamase production resulting in transformed host cell phenotypic resistance to ampicillin). Plasmid pKN402-derived plasmids including marker genes include, e.g., the ampicillin-resistance conferring plasmid pKN403 (DSM Accession No. 1229), plasmid pKN404 conferring streptomycin resistance, and plasmids pMOB45 and pMOB48 [10.5 Kb and 9.5 Kb, respectively, see Bittner, et al., Gene, 15, pp. 319-329 (1981)] which confer chloramphenicol and/or tetracycline resistance. With the insertion of even a moderately-sized exogenous gene with a functionally associated promoter/regulator sequence the plasmid size could easily extend to 11 or 12 Kb. Large plasmid size and attendant host cellular energy drains upon runaway replication result in significant problems in view of the relatively high "basal" copy numbers and the enormously high "runaway" copy numbers for a pKN402 and many of its derivatives. U.K. Pat. No. 1,557,774, for example, reports 50 plasmid copies per cell (a "copy number" of 25) for pKN402-tranformed E.coli at 30.degree. C. and amplification up to about 5000 plasmid copies per cell upon shift in cell culture temperature to 40.degree.. While such high copy numbers are advantageous in the sense of favoring extensive mRNA transcription of exogenous genes, much of the total cellular energy is wasted in DNA replication and mRNA transcription of DNA sequences having little, if any, consequence to the goal of exogenous gene expression. Depletion of cellular energy resources, of course, has a direct and unfavorable influence on the rate of mRNA translation into desired protein products.
The problems associated with relatively high basal copy numbers of common temperature sensitive runaway mutant plasmids take on even greater significance upon consideration of potential exogenous gene product toxicity because even at low ("permissive") temperatures the high rates of transcription and translation events can provide quantities of gene product which interfere with optimal host cell growth in culture. Manipulations geared toward reducing the basal and amplified (elevated temperature induced) copy number of runaway mutant plasmids have generally resulted in either loss of temperature sensitivity characteristics or loss of the capacity for maintenance of the plasmids in host cells. See, e.g., Hashimoto-Gotoh, et al., Gene, 16, pp. 227-235 (1981). Of interest to the background of the present invention is the independent notation of the existence of "partition" DNA sequences in pSC101-derived plasmids, which sequences reportedly facilitate stable plasmid inheritance. See, Meacock, et al., Cell, 20, pp. 529-542 (1980).
A partial attempt to deal with the problem of toxic gene product "leakage" occasioned by high basal copy numbers has taken the form of incorporation of structural gene transcription promoter DNA sequences functionally associated with "strong" repressor (operator) sequences which allow for highly selective chemical or, preferably, thermal control of exogenous gene expression. See, e.g., Sninsky, et al., U.S. Pat. No. 4,374,927; Sninsky, et al., Gene, 16, pp. 275-286 (1981); Remaut, et al., Gene, 22, pp. 103-113 (1983).
The use of "strong" promoter sequences in pKN402-derived plasmids, of course, occasions corresponding potential losses in overall efficiency of gene expression owing to the lack of correspondingly strong mRNA transcription termination sequences. In the absence of such sequences, mRNA transcription of exogenous genes will ordinarily be accompanied by a "read through" into adjacent DNA sequences with corresponding cellular energy drains, possible significant interference with exogenous gene mRNA binding to ribosomes (owing to "oversizing") and, in the case of the temperature sensitive runaway mutants, possible interference with proper transcription of DNA sequences essential to autonomous replication of the plasmid and the runaway characteristic.
Another disadvantage of pKN402-derived plasmids has been the general lack of unique restriction endonuclease enzyme recognition sites which would allow ready incorporation of exogenous gene sequences. Where present, such unique sites are frequently at positions intermediate a marker gene, effectively necessitating plasmid constructions involving two selectable phenotypic marker genes to allow for both exogenous gene insertion and verification of host transformation. See, Hashimoto-Gotoh, et al., supra, but cf, Remaut, et al., supra, discussing insertion of a "multilinker sequence" in a 7 Kb pKN402-derived plasmid designated pCP3.
Despite substantial efforts at modification of the originally isolated runaway mutants, there continues to exist a need in the art for small-sized, autonomously-replicating, stably-maintained, selectable, circular DNA plasmids having temperature sensitive copy number mutant replication characteristics. Optimal plasmids of this description would have relatively low basal copy numbers (in the range of 1 to 20 and preferably 1 to 10) as well as relatively low amplified, temperature elevation-induced, copy numbers (in the range of 100 to an unlimited number and preferably 100 to 300) and would thereby avoid both excessive cellular energy drains during maintenance and runaway condition induction and premature "leakage" of exogenous gene products into transformed host cytoplasm. Optimal plasmids would include DNA sequences operative to provide for ready incorporation of exogenous genes without interfering with the function of selectable marker genes and would also provide DNA sequences operative as mRNA transcription terminators. The latter DNA sequences would be provided at a locus wherein they could function to terminate transcription of inserted exogenous genes, especially those under the control of strong promoter DNA sequences which might optionally be provided in such optimal plasmids.